Functionally diverse proteins are generated by alternative splicing of primary transcripts in differentiating oligodendrocytes. PLP and DM20 are generated through the alternative selection of competing 5'splice sites in exon 3. As PLP becomes the predominant isoform, the PLP/DM20 ratio increases in differentiated oligodendrocytes (OL) versus progenitors (OPC). Mutations that alter the ratio of PLP to DM20 cause neurological disorders in humans [Hobson et al., 2006;Hobson et al., 2002]. One of these mutations is a deletion of a G-rich intronic enhancer (ISE) of the PLP 5'site. In the preliminary studies, we show that exon 3B contains sequences that regulate the PLP/DM20 ratio. A G-rich sequence (M2) is an enhancer of DM20 5'site, while the other exonic sequences enhance the PLP 5'site. Although both are G-rich, the ISE and M2 are functionally distinct. A number of hnRNP's bind to the ISE and M2 and some of them are down regulated in OL versus OPC. We hypothesize that M2 enhances the DM20 5'site in OPC, while the ISE favors the PLP 5'site in OL as a result of decrease in hnRNP's and changes in the balance of general and cell-specific factors. Other exon 3B sequences favor the PLP 5'site and are both general and cell-specific. In Aim 1, we will characterize the ISE's function within the full context of the PLP gene in the developing nervous system of a novel knockin mouse that carries a deletion of the ISE. The cell-specific and differentiation-dependent function of the ISE will be elucidated in the brain, nerves and non-glial tissues. In Aim 2, we will characterize the role of M2 in controlling the PLP/DM20 ratio in oligodendrocytes and non-glial cells by mapping the contribution of the G-sequences and flanking sequences to controlling the PLP/DM20 ratio. The proteins that bind to M2 and to ISE will be identified in biochemical studies and their expression will be examined in OPC and OL. In Aim 3 we will examine enhancers of the PLP 5'site and define their role in general, cell-specific and differentiation-dependent regulation of PLP/DM20 ratio. In Aim 4 we will examine the function of hnRNP's in controlling the PLP/DM20 ratio with knock down studies by RNAi. These studies have broad relevance to oligodendrocyte differentiation, generation of transcript diversity, and alterations of splicing that causes inherited disorders of myelin.